Genetic abnormalities are found in the majority of spontaneously aborted fetuses. In addition to inherited genetic defects, somatic mutation of the zygotic genome can also contribute to abnormal fetal development. Somatic mutation of critical genes may lead to embryonic lethality, or result in small clones of cells contributing to birth defects or disease susceptibilities later in life. This project focuses on a subset of a group of twelve heritable zebrafish mutations that cause increased frequencies of somatic mutation in embryos. The goal of this study is to identify the genes that have been mutated in these zebrafish strains. By studying genes that normally function to prevent somatic mutations during embryonic development in this model organism the investigator will further the understanding of how the same genes function in human embryonic development to prevent birth defects and disease. Two zebrafish genomic instability lines, known as the gin mutants, are the subject of this project. When homozygous, these recessive mutations cause increased frequencies of somatic mutations that are detectable using an assay for mosaic pigmentation of the embryonic retinal epithelium. These two gin mutations also show interesting effects when combined as double heterozygotes, indicating that they are mutations in genes with either interacting gene products, or that function in a common pathway. However, the individual genes that have been mutated in the gin fish have not yet been identified. Preliminary mapping experiments have identified an interval on Linkage Group 4 for one gin mutation, and an interval on Linkage Group 18 for the other gin mutation. A set of candidate genes within each of these intervals has been found by comparing the markers used for genetic mapping with the most recent genomic DNA sequence from the Danio rerio Sequencing Group at the Sanger Institute. The specific aims of this project are to 1) test the candidate genes for embryonic expression in wild-type embryos;2) knock-down and rescue experiments using candidate genes in embryos for genomic stability/instability activity;and 3) clone the most likely candidate genes from both wild-type and gin embryos to determine if they are the actual gin genes. Identification of the gin genes is critical, and will be the basis for future experiments exploring the mechanisms and potential genetic interactions that cause somatic mutations during development. PUBLIC HEALTH RELEVANCE: The investigator is studying a unique set of genetic strains of the small freshwater zebrafish, which show increased incidence of mutations from the earliest stages of embryogenesis. The goal of this project is to identify the genes responsible for these changes in two of the zebrafish mutant lines. By studying genes that cause somatic mutations in this model organism, she hopes to increase our understanding of human embryonic development, birth defects and disease.